Our long standing interest in cutaneous dendritic cell physiology continues. Recently developed mice that are constitutively, or that can be made conditionally, Langerhans cell-deficient allow definitive studies of Langerhans cell function and development to be carried out. One of these strains of mice and a variety of monoclonal antibodies allowed us to conclusively identify at least 3 distinct subsets of dendritic cells in skin and to begin to characterize lineage relationships between them. In addition, we have utilized Langerhans cell-deficient mice to identify an unexpected role for these cells in antibody forming responses. Immunization of transgenic mice at a time when they are Langerhans cell deficient via gene gun led to selective attenuation of the IgG1 isotype response. Since IgG1 formation is thought to be IL-4-dependent, this suggests that Langerhans cells may be specialized to present antigen to Th2 cells. How this might occur remains to be determined. These findings are described in a paper that was published in the Proceedings of the National Academy of Sciences in 2009. In ongoing studies, we are using alternative immunization strategies to discern mechanisms that allow Langerhans cells to regulate responses to topically applied protein antigens. The above studies have provided data that has resulted in development of new hypotheses regarding Langerhans cell ontogeny. We have examined the possibility that locally secreted influences that may be keratinocyte-derived may be important regulators of Langerhans cell differentiation. Both in vitro and in vivo approaches were utilized. Results obtained via these studies implicate Wnt-signaling in Langerhans cell development. Relevant findings have been published in the Journal of Investigative Dermatology in 2011. Collaborative studies of dendritic cells and their products in experimental cutaneous leishmaniasis have also continued and results continue to inform our understanding of dendritic cell function in this murine model of an important human disease. Recent results implicate the cytokine IL-17 and neutrophils in Leishmania pathogenesis in susceptible mice. It is anticipated that these insights will promote development of a vaccine that will attenuate the burden of this world-wide health problem. A publication describing these findings was published in the Journal of Immunology in 2009. In additional collaborative studies, the ability of recombinant Leishmania proteins containing the TAT protein transduction domain to serve as vaccine candidates in murine experimental cutaneous leishmaniasis has been tested. These studies indicates the ability to prime CD8 T cells correlates with disease protection and/or attenuation, and the findings were published in the Journal of Investigative Dermatology in 2010. Follow up studies utilizing IL-1 receptor- and IL-1 receptor antagonist (IL-1RA)-deficient mice have additionally characterized the involvement of these entities in the experimental cutaneous leishmaniasis in mice. These results were published in manuscripts appearing in Experimental Dermatology and the Journal of Investigative Dermatology in 2011. An additional collaboration involves Dr. Maria Morasso and her investigative group in NIAMS. Dr. Morasso has developed a novel mouse in which the transcription factor Dlx3 is selectively deleted in keratinocytes. This results in a barrier defect, production of a variety of chemokines and inflammatory mediators by keratinocyes and subsequent development of a systemic inflammatory syndrome that is characterized by prominent IL-17 production. We are playing a major role in the characterization of the inflammation that occurs in these mice. The initial description of the phenotype of these mice appeared in the Proceedings of the National Academy of Sciences in 2011 and additional studies are ongoing. The goal of these studies is to understand the mechanism(s) by which lack of expression of a transcription factor (Dlx3) in keratinocytes results in an inflammatory condition that shares some features with the human skin disease psoriasis. Using neutralizing monoclonal antibodies, we have identified cytokines that are particularly important in this mouse models and leukocytes that produce them. We are in the process of of elucidating the mechanism(s) by which loss of a transcription factor in epidermal keratinocytes leads to production of relevant cytokines by cells of interest. The laboratory has long been interested in elucidating effects of the cytokine TGF beta on epidermal Langerhans cells and dendritic cells. In a recent collaboration with Dr. Adam Glick and co-workers, we helped elucidate effects of over expression of TGF beta in skin on dendritic cell homeostasis in mice. A surprising result was that TGF beta over expression mobilized cutaneous dendritic cells and enhanced contact hypersensitivity reactions. These results and supporting data was published on the Journal of Investigative Dermatology in 2012. A final collaboration involves Dr. Keisuke Nagao and his colleagues at Keio University in Tokyo. In continuing studies that he initiated in my laboratory, Dr. Nagao and co-workers have determined that mouse epidermal Langerhans cells can derive from several different precursors in different settings and, surprisingly, that terminal hair follicles serve as portals of entry into epidermis, very likely reflecting production of selected chemokines by subpopulations of hair follicle keratinocytes. These findings have recently been published in Nature Immunology.